Delivering substances to invertebrate organisms

ABSTRACT

Particles ( 10 ) for controlling, treating, feeding of aquatic invertebrate filter feeding organisms such as mussels, clams, oysters and insect larvae comprising a biologically active ingredient ( 13 ) embedded within or surrounded by an innocuous carrier material ( 12 ). The organism effectively concentrates the active ingredient within its body by feeding on the particles, thereby reducing the total amount of active ingredient required to be used.

[0001] The present invention relates to a composition and method for usein the delivery of a desired substance to invertebrate organisms. Theinvention is particularly applicable, but not restricted to, aquaticfilter feeders such as bivalve molluscs (for example, mussels and clams)and insect larvae and may be used to deliver any desired substancehaving biological activity in the organism concerned, for example one ormore toxic agents, growth promoters, nutrients, anti-parasitic agents,growth controllers, or reproduction promoters/inhibitors.

[0002] The present invention is concerned with the manipulation of anyaspect of an invertebrate's physiology, growth, reproduction, resistanceor vulnerability to disease or parasitic infestation or behaviour,including killing invasive invertebrate organisms by delivering aneffective amount of a toxic agent thereto. “Manipulation” is to beinterpreted also as restoring normal function and/or growth in anenvironment which would otherwise not support normal function and/orgrowth, for example a nutrient depleted environment.

[0003] Toxic agents may be used which have the effect of killing,weakening, debilitating or indeed inducing any effect which enables thepest to be displaced or more easily removed from the area or environmentbeing controlled.

[0004] Conventionally, it is known to address the problem ofinfestations of bivalve mussels which adhere by means of their byssusthreads or “beards” to hard substrates, such as power station coolingwater intakes and associated auxiliary equipment, by injecting anaqueous solution of toxin into the water stream. Suitable toxins includechlorine-based agents such as sodium hypochlorite at a concentration ofapproximately 3 ppm.

[0005] It has been known for some time that simply putting the desiredsubstance, for example a toxin, into the water in which bivalvemolluscs, particularly mussels, are feeding can cause the mussels toclose their shells and cease feeding, whereupon the substance has to becontinually added to the water for about three weeks until the musselsare forced to resume feeding. This means that there is a considerableand undesirable amount of water pollution by the toxin.

[0006] Various attempts have been made to find a more environmentallyacceptable way of delivering a substance such as a toxin to bivalvemolluscs. For example, in U.S. Pat. No 5,252,330 to The University ofToledo, Zebra mussels (Dreissena polymorpha) are controlled bycontacting the mussels with effective lethal amounts of an aqueouschemical treating medium comprising molluscicidally effective portionsof the berry from Phytolacca dodecandra (also known as Endod) whichcontains a toxin (“Lemmatoxin”) having the structural formula disclosedin British Patent No 1,227,417. When used as a treating agent in water,the Phytolacca treatment is preferably incubated to increase itschemical activity.

[0007] However, even such a plant-based and biodegradable toxin cancause considerable environmental damage and will kill organisms otherthan the target organism.

[0008] Accordingly it is one of the aims of the present invention toaddress problems with known compositions and methods of deliveringsubstances to invertebrate organisms, whether referred to herein orotherwise.

[0009] The Applicants have surprisingly found that one or more desiredsubstances may be targeted to the intended organisms (particularlymussels and clams) more efficiently if the organisms can be induced tocontinue ingesting the substance, thereby allowing a smaller totalquantity of the substance to be used to achieve the desired effect,thereby minimising the environmental impact of the substance.

[0010] According to a first aspect of the present invention there isprovided particles for ingestion by an invertebrate organism, saidparticles including at least one ingredient having desired biologicalactivity in the organism when ingested by the organism and at least onecarrier material, characterised in that the active ingredient is solid,toxic to the organism, and coated with or encapsulated within a watersoluble carrier material.

[0011] Conveniently the above ingredient is embedded throughout thecarrier material, or alternatively the active ingredient may be coatedwith or encapsulated within (for example by known microencapsulationtechniques) the carrier material.

[0012] Preferably the organism is an aquatic invertebrate organism, forexample a filter feeder such as a bivalve mollusc. Examples of bivalvemolluscs to which the invention may be applied include mussels,particularly Zebra mussels, clams, particularly the Asian clam(Corbicula Fluminea), and oysters. Examples of other aquaticinvertebrates to which the invention may be applied include insectlarvae such as blackfly larvae. In tropical Africa the blackfly Simulindamnosum is the vector of onchoceriasis (river blindness) as well asreducing yields in cattle populations due to the blood-sucking habit ofthe adult flies which prevents efficient foraging. The only currentmethod of blackfly control is to treat the water with DDT(dichlorodiphenyltrichloroethane) which presents major environmentalrisks.

[0013] By “biological activity” is meant any effect, or combination ofeffects, on any aspect of the organism's physiology, growth,reproduction, disease or parasite resistance or vulnerability, orbehaviour, and includes causing or hastening the death of the organismeither directly or indirectly. An ingredient having such biologicalactivity in the organism will hereinafter be referred to as “the activeingredient” and includes substances, such as nutrients, which restorenormal function and/or growth, in an environment which would otherwiseinhibit or restrict said normal function and/or growth, for example dueto insufficient, or complete lack of, essential nutrients.

[0014] Particularly, it is preferred that the effect is a physiologicaleffect.

[0015] To render the particles ingestible, firstly the particle size ischosen to be suitable for ingestion by the target organism. For example,for bivalve molluscs such as mussels the mean particle size ispreferably in the region of 1 to 200 microns or more in diameter, morepreferably between 2 to 150 microns in diameter.

[0016] Zebra mussels are filter feeders, filtering around 1 gallon ofwater a day and selecting for ingestion particles up to 200 microns indiameter. The filtering structures are the large fleshy gills that lieon each side of the body, within the mantle cavity and these gills arecovered by different types of cilia. The frontal cilia on the outer gillface beat towards the marginal groove which runs along the ventral freeedge of the gill—coarse particles in excess of 200 microns pass down thecrests of the gill surface and are excluded from the marginal groove,whereas smaller particles are directed along the groove and arrive atthe labial palps. A proportion of the particles arriving at the labialpalps are passed into the mouth.

[0017] For example, by providing the active ingredient in the form ofparticles which are compatible with this selective feeding mechanism andwhich are not detected by the organism as being toxic, the activeingredient is more effectively taken up by and will be concentrated inthe target organism.

[0018] Preferably, the carrier material is not only innocuous for thetarget organism but is preferably also nutritious for and/or attractiveto the target organism, thereby enhancing the ingestion of theparticles.

[0019] In the preferred embodiment, the active ingredient is provided asa core surrounded by a coating of innocuous and/or nutritious and/orattractive carrier material around said core.

[0020] Alternatively, the treatment medium may be manufactured bymicroencapsulation techniques such as complex coacervation which iscapable of producing microcapsules of between 10 and 800 microns indiameter.

[0021] Two possible encapsulation techniques are fluidised bed spraycoating and spray congealing.

[0022] Using fluidised bed spray coating it has been possible to produceencapsulated particles in the correct size range, for example consistingof 75% by weight palmitic or stearic acid and 25% potassium chloride orpotassium permanganate (the fluidised solid material was 43±10 μm or98±10 μm).

[0023] Using spray congealing techniques, fluid milled potassiumchloride particles of less than 10 μm diameter were suspended inpalmitic acid and the dispersion sprayed to produce particles in therange 10 to 100 μm.

[0024] If embedded within the ingestible particle, the active ingredientis preferably provided in the form of much smaller “sub-particles”,evenly distributed throughout the ingestible particle.

[0025] If encapsulated within an outer ingestible coating, the activeingredient preferably comprises a core of between about 40 to 60 micronsin diameter. The thickness of the coating is preferably between 5 and 40microns, more preferably about 10 microns.

[0026] Where the active ingredient is toxic to the target organism, thepreferred toxin is an inorganic solid such as potassium chloride whichinduces heart attack in certain invading species such as mussels andclams. The potassium chloride is preferably provided in the form ofcrystals of appropriate size. An alternative to potassium chloride ispotassium permanganate.

[0027] Alternative toxins include saponins, more preferably “Lemmatoxin”derived from Endod berries, or a synthesised form of its activeingredients. A combination of two or more different toxins, or a toxincombined with one or more further active ingredients, may be used.

[0028] The particles are preferably at least partially resistant towater, such that they may be suspended in water without undue leachingof the active ingredient into the water, for example they should retainat least 75% of the active ingredient when immersed in water for severalhours.

[0029] The carrier material may conveniently be manufactured fromstarch, such as potato starch, which may be provided in the form of apaste, or chocolate which is preferably 70% cocoa solids. Alternativeingestible carrier materials suitable for making the particles includebeeswax, fatty acids such as palmitic acid, stearic acid, oils, fats andwaxes or derivatives thereof, or dried plankton, e.g. driedphytoplankton or dried zooplankton. Clearly, the nature of theingestible substance will vary dependent upon the target organism, andis preferably a material which is both nutritious and attractive to theparticular target organism. Any combination of two or more of theaforesaid substances may be used. A carrier material including orcomprising one or more fatty acids is particularly preferred as it formsa hard coating and is slightly soluble in water, thus allowing theparticles to remain active for their residence time in the water. Forexample, a 100 μm stearic acid particle will dissolve in stagnant waterin 124 hours.

[0030] Preferably the particles have neutral buoyancy in freshwater,corresponding to an optimum density for the particles of 1 g/cm³, whichcan be achieved using, for example 26% KCl in palmitic acid or 11% KClin stearic acid. The hydrophobicity of such particles inhibits dispersalin water but dispersal may be facilitated by the is use of a surfactantsuch as sodium palmitate at around 1 wt % of the particles.

[0031] According to a second aspect of the present invention there isprovided a method of inducing an invertebrate organism to ingest asubstance having desired biological activity in the organism wheningested by the organism, the method including introducing into theorganism's environment particles ingestible by the organism andincluding the substance and at least one carrier material, wherein thesubstance is solid and coated with or encapsulated within the carriermaterial and wherein the substance is toxic to the target organism.

[0032] The method preferably includes the step of contacting theorganisms with the particles for at least 4 hours, more preferably forbetween 4 and 8 hours.

[0033] According to this second aspect of the present invention there isfurther provided a method of concentrating, to a biologically activeconcentration within an invertebrate organism, at least one substance,the method comprising providing in the organism's environment particlescontaining the substance and at least one carrier material, theparticles being ingested by the organism such as to effect saidconcentration of the substance, wherein the substance is solid andcoated with or encapsulated within the carrier material and wherein thesubstance is toxic to the target organism.

[0034] According to a third aspect of the present invention there isprovided a method of preventing cessation of feeding in an invertebrateorganism which would otherwise occur due to the presence of a substancein the organism's environment, the method including the step ofproviding the substance in the form of particles ingestible by theorganism, said particles also including a carrier material, wherein thesubstance is solid and coated with or encapsulated within the carriermaterial and wherein the substance is toxic to the target organism.

[0035] Thus, because the organism continues to feed, the substance iseffectively concentrated within the organism and reduces the totalamount of substance required to be added to the organism's environmentto have the desired effect on the organism.

[0036] According to a fourth aspect of the present invention there isprovided a method of controlling an invasive or potentially invasivepopulation of invertebrate organisms, comprising feeding said populationwith an effective amount of a composition comprising particlescontaining a carrier material and at least one active ingredient,wherein the active ingredient is solid and coated with or encapsulatedwithin the carrier material and wherein the active ingredient is toxicto the target organism.

[0037] By “effective amount” it is meant sufficient to have a desiredbiological effect on the population or on individuals within thepopulation.

[0038] Preferably, said active ingredient is toxic to the targetorganism and the effective amount is a sufficient amount to kill asignificant number of individuals within the population, or to reducethe tendency of the population to is become or remain invasive, forexample in the case of mussels, their tendency to adhere to each other(aggregation) and/or to the underlying substrate.

[0039] Where individual organisms are killed, death may occur eitherduring the treatment of the present invention or sometime aftertreatment as a direct or indirect result of the treatment.

[0040] According to a fifth aspect of the present invention there isprovided a method of treating water containing invertebrate organisms,comprising adding to the water particles containing at least one carriermaterial and at least one active ingredient, said particles beingingestible in an effective amount by the invertebrate organisms, whereinthe active ingredient is solid and coated with or encapsulated withinthe carrier material and wherein the active ingredient is toxic to thetarget organism.

[0041] Preferably, the particles, or at least the carrier material,are/is at least partially resistant to water such that the activeingredient is inhibited from leaching into the water for sufficient timefor an effective amount of the composition to be ingested by the targetorganisms.

[0042] According to a sixth aspect of the present invention there isprovided a method of controlling parasites or symbionts in or on a hostinvertebrate organism, the method comprising feeding the host organismwith particles including at least one carrier material and at least oneactive ingredient having biological activity in the host and/or in theparasite or symbiont, wherein the active ingredient is solid and coatedwith or encapsulated within the carrier material and wherein the activeingredient is toxic to the target organism.

[0043] According to an seventh aspect of the present invention there isprovided a method of rendering an environment suitable for the farmingof invertebrate organisms, the method comprising adding to theenvironment particles containing at least one carrier material and atleast one active ingredient, said particles being ingestible by theorganisms, wherein the active ingredient is solid and coated with orencapsulated within the carrier material and wherein the activeingredient is toxic to the target organism.

[0044] Preferably, said active ingredient is a nutrient or growthfactor.

[0045] According to a eighth aspect of the present invention there isprovided a food product for the farming of invertebrate organisms, thefood and product comprising particles ingestible by the organisms havingat least one active ingredient and at least one carrier material,wherein the active ingredient is solid and coated with or encapsulatedwithin the carrier material and wherein the active ingredient is toxicto the target organism.

[0046] Preferably, the particles include several nutrients relevant tothe nutritional requirements of the organisms to be farmed.

[0047] Any feature of any aspect of any invention or embodimentdescribed herein may be combined with any other feature of any aspect ofany invention or embodiment described herein.

[0048] Embodiments of the present invention will now be described, byway of example only, with reference to the accompanying drawings, inwhich:

[0049]FIG. 1 illustrates a particle having an active ingredienthomogeneously embedded therein, in accordance with the presentinvention,

[0050]FIG. 2 illustrates a particle having a core of active ingredientsurrounded by an edible coating,

[0051]FIG. 2a illustrates a method of producing particles with astarch-based carrier material,

[0052]FIG. 3 illustrates the results shown in table 2 in the form of agraph.

[0053]FIG. 4 is a graph of time taken for three Zebra mussels to diewhen exposed to different concentrations of Endod.

[0054]FIG. 5 is a graph of mean time for Zebra mussels to die withdifferent concentrations of Endod.

[0055]FIG. 6 comprises two graphs of mean time for Zebra mussels to diewhen exposed to 30 mg/l Endod that had been allowed to decay in waterfor differing numbers of days. Endod was decayed at 15° C. and 40° C.(Asterisks denote experiments that were run where no Zebra mussels died,^(a) denotes an experiment where a single Zebra mussel died.

[0056]FIG. 7 is a graph of the proportion of Zebra mussels that wereobserved to be open or closed when placed in filtered pond water orfiltered pond water with lethal doses of Endod.

[0057]FIG. 8 is a photograph of a Zebra mussel which has been exposed towater containing a suspension of oil paint in vegetable oil. The arrowindicates the gut, which has become stained with the ingested particlesof pigment.

[0058]FIG. 9 is a graph of mean numbers of mussels alive duringExperiment 8.

[0059]FIG. 10 is a graph of the mean lengths of remaining mussels atintervals during Experiment 8.

[0060]FIG. 11 is a comparison of the mean sizes of survivors offatalities after Experiment 8.

[0061] FIGS. 12 to 16 are scanning electron micrographs (SEMs) ofparticles in accordance with the present invention.

[0062]FIG. 17 is a series of photomicrographs of particles from twodifferent samples of particles during size fractionation.

[0063] Each of the particles, or at least a substantial percentagethereof, making up the composition and for use in the method of thepresent invention may comprise a particle 10 (see FIG. 1) of carriermaterial 12 such as chocolate (a substance edible by the targetorganism, for example the Zebra mussel (Dreissena polymorpha)), withinwhich is embedded much smaller “sub-particles” 13 of an activeingredient, in the case of the Zebra mussel, potassium chloride KClcrystals. The preferred size of the particle 10 is about 100 microns indiameter.

[0064] Other preferred carrier materials include dried phytoplankton ordried zooplankton.

[0065] A more preferred type of particle 10′ is that shown in FIG. 2 inwhich a core 14 of a single KCl crystal of about 53 micron diameter,surrounded by a carrier material (e.g. chocolate) coating 16, the wholebeing then ground back down to approximately 90 micron diameter.

[0066]FIG. 2a illustrates an alternative method of producing particlesaccording to the present invention, in which starch powder 20 is mixedwith distilled water 22 (or Kcl solution, for a toxic particle),extruded through a die 24 to make flake 26 which is then passed withzirconia balls through a pulverizer 28 and passed through a 75 μm sieve30 to produce particles of diameter ≦75 μm.

Experiment 1

[0067] The toxicity of KCl alone to Zebra mussels is illustrated inTable 1 below, which gives the results of the following experiment:

[0068] 1) A series of pots where filled with 20 ml of distilled waterplus a specified mass of KCl crystals, ground to less than 53 microns.The masses of KCl were chosen to represent the total amount of KCl thatwould be ingested by a mussel filtering 1 litre of water per day.

[0069] 2) One mussel was placed in each pot (it was assumed that themussel would continuously re-filter the limited amount of solution inthe pot and thus ingest the required amount of KCl.

[0070] 3) The mussels were observed at intervals during the followingtwo days, and their state noted (alive, dead or closed, i.e mussel stillalive but temporarily ceased feeding). TABLE 1 Toxicity of differentmasses of KCl to Zebra mussels over two days. Date 17/03/99 18/03/9918/03/99 19/03/99 Mass of KCl Time (mg) 16:30 11:30 13:30 16:00 0(control) Alive Closed Alive Dead 50 Alive Dead Dead Dead 25 Alive DeadDead Dead 10 Alive Alive Closed Dead  5 Alive Alive Alive Alive  1 AliveAlive Alive Alive   0.5 Alive Alive Closed Alive   0.1 Alive Alive AliveAlive

[0071] As can be seen from the above table, using KCl alone is noteffective over the test period at lower concentrations. The aim of theinvention is to reduce the concentrations down to less than 1% of bulkconcentration, which corresponds to the 0.5 mg mass in Table 1, whichclearly would not have the desired effect of controlling the musselsusing KCl alone.

Experiment 2

[0072] The toxicity of an alternative toxin was also investigated andcompared with that of KCl. In an another experiment, the results ofwhich are shown in Table 2 and illustrated in the graph of FIG. 3, Endodwas ground to approximately 75 micron particle size which was then fedto a sample group of five mussels and was shown to achieve totalmortality of the mussels at low Endod concentrations over a period ofapproximately 5 days. TABLE 2 toxicity of Endod to Zebra musselscompared with KCl Time for mortality Mass of toxin of all mussels Toxin(mg) (hours) Endod 0 — Endod 0.5 102 Endod 7.5 30.5 Endod 25 23 Endod100 102 (80% mortality) Potassium Chloride 100  8 (60% mortality)

[0073] One particular advantage of Endod as a toxin is its ability toachieve latent mortality (death after Endod has left the system) inZebra mussels. Powder from dried Endod berries is lethal to Zebramussels and those that did not die failed to reaggregate and reattach(Lemma et al, 1991). A short dose for 4-8 hours provides 50% mortalityplus continuing resultant mortality. Endod can also be easily adsorbedusing activated charcoal beds, but does not as yet have regulatoryapproval for its use in cooling water systems. Toxicity of Endod toother, non target organisms compared with Zebra mussels is shown inTable 3, which gives both the 48 hour LC₅₀ value as well as the 95%confidence interval (the 48 h-LC₅₀ is the concentration of toxinrequired to induce mortality in 50% of the population within 48 hours).TABLE 3 Toxicity of Endod to target and non target organisms (taken fromWaller, Rach, Cope and Marking 1993: Toxicity of Candidate Molluscicidesto Zebra Mussel and Selected Non-target organisms). 48-hour LC₅₀ and 95%confidence interval Zebra Mussel Rainbow Channel Threehorn 20-25 TroutCatfish Wartyback mm 5-8 mm 0.8-1.2 g 0.8-1.2 g 30-50 mm KCl 150 1471610 720 >2000^(a) 129-  132-163    1223-2119   588-882  175 Endod>10^(a)  9.51   1.31  1.60  >30^(a) 8.50-10.65  1.12-1.53  1.23-2.08

Experiment 3

[0074] Lethal concentrations of Endod were identified in the followingexperiment.

[0075] Six pots, each containing three Zebra mussels of approximately 2cm length, were filled with 100 ml of filtered pond water. Each pot wascontinually aerated and held at a constant 15° C. Mussels were allowedto acclimatise for two hours before different masses of ≦75 μm Endodpowder (from dried berries) was added to five of the pots (0.002 g,0.005 g, 0.01 g, 0.03 g, 0.1 g). The sixth pot served as a control. Thenumber of mussels that were dead in each pot was monitored regularlyover 4.5 days. The observations are shown below in Table 4 and theseresults are presented graphically in FIGS. 4 and 5.

Results

[0076] TABLE 4 Establishing ball park lethal dose of Endod required. O =Open C = Closed D = Dead 2 mg Time Day Time Endod/litre 5 mg 10 mg 30 mg100 mg Control (hrs) Wed  3:00 pm 1O 2C 3C 3O 3C 3C 3C 0.5  3:30 pm 2O1C 3C 3C 3C 3C 3O 1  4:00 pm 2O 1C 2O 1C 3C 3C 3C 3O 1.5  4:30 pm 2O 1C3O 1O 2C 3C 3C 3O 2  5:30 pm 3O 2O 1C 1O 2C 3C 3C 3O 3  6:00 pm 3O 3C 3C3C 3C 3O 3.5  6:30 pm 3O 2O 1C 3C 3C 3C 3O 4  7:30 pm 2O 1C 1O 2C 3C 3C3C 2O 1C 5 10:30 pm 2O 1C 1O 2C 1O 2C 1O 2C 3C 2O 1C 8 Thur  9:00 am 2O1C 1O 2C 3C 3D 3D 2O 1C 18.5 12noon 1O 2C 1D 2C 3C 3D 3D 2O 1C 21  2:30pm 1O 2C 1D 1O1C 3C 1O 2C 23.5  3:00 pm 1O 2C 1D 2C 1D 2C 1O 2C 24  3:30pm 3C 2D 1C 1D 2C 1O 2C 24.5  4:00 pm 1O 2C 2D 1C 1D 2C 2O 1C 25  4:30pm 3C 2D 1C 2D 1C 2O 1C 25.5  5:30 pm 1O 2C 2D 1O 2D 1C 2O 1C 26.5  9:00pm 3C 2D 1C 2D 1C 1O 2C 30 10:00 pm 1O 2C 2D 1C 2D 1C 2O 1C 31 Fri  9:00am 3C 2D 1C 2D 1C 3O 42 10:30 am 1O 2C 2D 1C 2D 1C 3O 43.5 11:30 am 3O2D 1C 2D 1C 2O 1C 44.5  3:00 pm 3O 2D 1C 3D 1O 2C 48 Sat 10:00 am 3D 3D3D 2O 1C 67 Sun  9:00 pm 1O 2C 3D 3O 102 Mon 10:00 am 2O 1C 3D 2O 1C 115Tues 10:00 am 3O 3D 3D 3D 3D 3C 139 10:00 pm 3O 2O 1C 151 Wed  9:00 am3O 1O 2C 162 11:00 pm 1O 2C 1O 2C 176 Thur 11:30 am 3C 3C 188.5  4:00 pm2O 1C 2O 1C 193 Fri  9:00 pm 2O 1C 3O 212 Sat  4:00 pm 2O 1C 3O 23110:30 pm 3C 1O 2C 237.5 Sun  9:00 pm 2O 1C 1O 2C 260 Mon  9:00 am 2O 1C1O 2C 272

[0077] No mussels died in the control pot, or with Endod at 2 mg/l(FIGS. 4 and 5). All mussels had died within 36 hours where the musselsexperienced 30 and 100 mg/l Endod and all mussels had died by 84 hoursat 5 mg/l Endod (FIG. 4). Mussels died most quickly at the highestconcentrations of Endod (FIG. 5).

Experiment 4

[0078] In this experiment it was assessed how quickly Endod biodegradesunder different temperature regimes. Once it had been established fromexperiment 3 that 30 mg/litre Endod was lethal to Zebra mussels, sixsets of three pots were filled with 100 ml filtered pond water and 0.03g ≦75 μm Endod powder and held at a constant 15° C. The Endod wasallowed to decay in the pots for 6, 4, 3, 2, 1 and 0 days after whichthe water was poured through filter paper and the residue resuspended in100 ml filtered pond water. This ensured that toxins associated with thebacterial breakdown of the Endod did not interfere with the experiment.Three Zebra mussels (approximately 2 cm length) were added to each pot,the pots held at a constant 15° C., and the mussels observed at regularintervals. At each inspection, the number of mussels that were dead wasrecorded. Observations were made over six hours or until all mussels haddied.

[0079] The experiment was repeated with Endod which was allowed to decayat a constant 40° C. over 3, 2.5, 2, 1.5, 1. 0.5 and 0 days. Oncemussels were added to the pots they were transferred to a constant 15°C. Observations were made over 33 hours. The observations are shown intables 5 and 6 and these results are shown graphically in FIG. 6.

Results AppenDix 1—Decay at 15C.

[0080] TABLE 5 Time(Days) Time(hrs) CONTROL 6 4 3 2 1 0 0 3C 3C 3C 3C 3C3C 2C 1O 3C 3C 3C 2C 1O 3C 3C 3C 2C 1O 3C 3C 2C 1O 2C 1O 3C 3C 23 2O 1C2O 1C 3O 2O 1C 1D 2C 1D 2C 3C 2O 1C 3O 1O 2C 2O 1C 1O 2C 1O 2C 1O 2C 2O1C 1O 2C 2O 1C 2O 1C 1O 2C 1O 2C 2O 1C 29 2O 1C 3O 2O 1C 3O 1D 2C 1D 2C2O 1C 2O 1C 3O 3O 1O 2C 3C 1D 2C 1O 2C 2O 1C 2O 1C 2O 1C 2O 1C 3C 1O 2C3C 45.5 3O 2O 1C 2O 1C 1O 2C 2D 1C 2D 1C 3C 3O 3O 2C 1O 1O 2C 3C 3D 3C3O 2O 1C 3O 3O 3C 1D 2C 1O 2C 50 2O 1C 1O 2C 3O 3O 2D 1C 2D 1C 2O 1C 3O1O 2C 3O 2O 1C 3C 3D 1O 2C 1O 2C 2O 1C 2O 1C 3O 3C 2D 1C 2O 1C 71 1O 2C2O 1C 1O 2C 1O 2C 3D 2D 1C 3O 3C 3C 3O 1O 2C 3C 3D 2O 1C 1O 2C 3C 2O 1C2O 1C 3C 2D 1C 2O 1C 75 1O 2C 1O 2C 3O 2O 1C 3D 3D 3O 1O 2C 3C 2O 1C 2O1C 3C 3D 3O 1O 2C 3C 2O 1C 3C 3C 2D 1C 3O 77.5 2O 1C 1O 2C 1O 2C 1O 2C3D 3D 2O 1C 1O 2C 3C 3O 2O 1C 3C 3OD 1O 2C 1O 2C 3C 2O 1C 1O 2C 3C 2D 1C1O 2C 78.5 3O 2O 1C 3O 3C 3D 3D 2O 1C 2O 1C 3C 2O 1C 1O 2C 1O 2C 3D 2O1C 2O 1C 2O 1C 2O 1C 3C 1O 2C 2D 1O 2O 1C 91 2O 1C 1O 2C 3O XXX 3D 3D 3O1O 2C 2O 1C 2O 1C 1O 2C 1O 2C 3D 3O 1O 2C 1O 2C 2O 1C 3C 2O 1C 2D 1C 3O93 2O 1C 3C 3O XXX 3D 3D 3O 1O 2C 1O 2C 1O 2C 1O 2C 1O 2C 3D 2O 1C 1O 2C1O 2C 2O 1C 3C 3C 2D 1C 3O 94 1O 2C 3C 3O XXX 3D 3D 1O 2C 1O 2C 3O 2O 1C3C 3C 3D 2O 1C 1O 2C 2O 1C 2O 1C 3C 3C 2D 1C 2O 1C 116 3C 1O 2C 1O 2CXXX 3D 3D 3C 3C 1O 2C 1O 2C 3C 3C 3D 3C 2O 1C 3C 1O 2C 3C 1D 2C 2D 1C 2O1C 122 3C 3C 2O 1C XXX 3D 3D 1O 2C 3C 1O 2C 2O 1C 1O 2C 3C 3D 1O 2C 1O2C 1O 2C 1O 2C 3C 3D 3D 3C 126 3C 2O 1C 3O XXX 3D 3D 3C 2O 1C 1O 2C 3O1O 2C 3C 3D 3C 2O 1C 3C 2O 1C 1O 2C 3D 3D 1O 2C 138 3O 1O 2C 1O 2C XXX3D 3D 3C 3C 3C 1O 2C 1O 2C 2D 1C 3D 1O 2C 3C 10 2C 3C 10 2C 3D 3D 10 2C146 20 1C 3C 20 1C XXX 3D 3D 10 2C 20 1C 3C 10 2C 10 2C 3D 3D 20 1C 102C 10 2C 10 2C 10 2C 3D 3D 10 2C

AppenDix 1—Decay at 40C.

[0081] TABLE 6 Time (Days) Time(hrs) 3 2.5 2 1.5 1 0.5 0 0 3C 3C 3C 3C3C 10 2C 3C 3C 3C 3C 3C 3C 3C 3C 3C 3C 3C 3C 10 2C 3C 3C 13.5 3C 3C 3C10 2C 10 2C 3C 30 3C 20 1C 3C 3C 3C 10 2C 3C 3C 10 2C 3C 3C 3C 3C 3C 3220 1C 10 2C 3D 3D 3D 3D 1D 2C 20 1C 10 2C 3D 3D 3D 3D 3C 10 2C 3C 3D 3D3D 3D 3C

[0082] Endod remained active up until two days, after which the Endoddid not kill any mussels. This result held true under both temperatureregimes (FIG. 6). At 15° C. mussels died more quickly with Endod thathad been held in water for only one day compared with two days. Musselsplaced with Endod which had been allowed to decay for 0 days (i.e. theEndod was suspended in water and then immediately filtered) resulted inonly one mortality from the six pots (eighteen mussels). No mussels diedin control pots.

Experiment 5

[0083] In this experiment Zebra mussels were tested to see if theyshowed this closing response in the presence of Endod.

[0084] At each inspection during experiment 4, it was recorded how manymussels were closed or were open and respiring normally.

Results

[0085] After 77.5 hours, all but one of the nine mussels exposed toEndod had died. Mussels exposed to Endod remained closed for almost theentire experimental period, while a large proportion of the mussels incontrol pots were open for the majority of time (FIG. 7).

Experiment 6

[0086] In this experiment it was tested whether novel, marked organicparticles were ingested by live Zebra mussels.

[0087] A suspension of marked organic particles was produced by mixingvegetable oil with red pigmented oil paint and stirring this intofiltered pond water. Approximately twenty Zebra mussels (lengthsapproximately 0.5-3 cm) were added to the suspension and left for 48hours, after which the mussels were killed and opened.

Results

[0088] A large proportion of the Zebra mussels were seen to be open atall times during the course of the experiment. On opening the mussels itwas clear that many had ingested the marked particles by the presence ofa red gut showing through the wall of the visceral mass (FIG. 8). It wasless clear to observe by eye the red guts of the smallest mussels,although dissection of the visceral mass often revealed the presence ofred particles within the gut lumen.

[0089] The results of experiments 3 to 6 provide strong support for thefeasibility of an encapsulated product. It has been shown that Zebramussels close-up in the presence of raw, non-capsulated toxins such asEndod and therefore an encapsulated product should reduce greatly theamount of toxin that would be required to induce mortality compared withsimply dumping toxins into the water. It has also been shown that novelorganic particles can be taken into the guts of Zebra mussels of allsizes.

[0090] Endod proved to be an effective toxin, inducing mortality atconcentrations >2 mg/l. In theory, this lethal concentration of Endodcan be reduced significantly if the toxin is encapsulated as proposed.It is promising that Endod biodegrades after two days in the watercolumn, irrespective of water temperature. This means that Endod willnot bioaccumulate in the ecosystem and therefore makes it a highlysuitable product to use in closed systems such as the Great Lakes. It isalso promising that Endod remains active at temperatures as high as 40°C., because this means that it will remain active within the heatedwaters of power stations.

Experiment 7

[0091] Two types of particles containing KCl and chocolate mixtures wereprepared. One type of particle comprised an edible chocolate substrateof about 100 micron diameter, within which was embedded a number of muchsmaller crystals of KCl (FIG. 1) whereas the other type of particlecomprised a single KCl crystal of about 53 micron diameter coated withchocolate and then ground back to about 90 micron diameter (FIG. 2).When these particles were suspended in water in which mussels were kept,three out of four mussels died within two days.

Experiment 8

[0092] A batch of particles (“Batch 7”) was manufactured by a fluidisedbed coating method. 98 μm KCl particles were passed through a 350 μmsieve with 1% silica to help fluidisation. 800 g of solids were sprayedwith palmitic acid (MP 61° C.) for 20 minutes at approximately 12 mlmin⁻¹. 400 g of the resulting solids were then further coated withpalmitic acid to finally produce particles containing between 20 and 25%wt of KCl.

[0093] 40 1-litre beakers were filled with 500 ml of tap water andaerated overnight in a temperature controlled room at 25° C. Ten largeZebra mussels were placed into each beaker, attempting to keep a similarsize range in all. Five different treatments were applied (such thateach had eight replicates);

[0094] 1. 5 g of (Batch 7) was added.

[0095] 2. 0.5 g of (Batch 7) was added.

[0096] 3. 0.05 g of (Batch 7) was added.

[0097] 4. 5 g of Palmitic acid alone was added.

[0098] 5. 1.15 g of KCl (the same amount by mass as in 5 g of Batch 7)was added.

[0099] Each treatment substance was thoroughly stirred into each beakerand aeration continued for the duration of the experiment. At reasonablyregular intervals, the number of dead mussels (as indicated by gape)were counted in each pot, before being removed and measured. This wascontinued until most were dead, or 72 hours had elapsed.

[0100] The results are shown in Tables 7, 8 and 9 below and representedgraphically in FIGS. 9, 10 and 11. Lines 1 to 5 correspond to treatments1 through 5 respectively. TABLE 7 Length of mussels dying over time whenexposed to 5 g of Batch 7 C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 Time(Hrs) 0 0.51 1.5 2 2.5 3 3.5 4 8.5 1 32 25 23 25 28 24 28 27 19 20 2 27 27 23 25 3227 30 34 23 24 3 30 28 28 29 33 28 30 32 26 28 4 31 26 29 29 28 30 23 3227 27 5 27 29 25 25 29 28 26 27 28 32 6 28 34 27 28 32 30 27 28 20 30 728 22 33 24 25 30 34 19 24 23 8 25 24 23 24 24 23 32 23 28 29 9 27 24 2528 27 26 32 26 27 23 10 28 25 25 32 28 27 27 27 32 11 26 26 29 33 30 3428 28 30 12 29 28 29 28 28 32 19 20 23 13 34 27 25 29 30 32 23 24 29 1422 22 28 32 30 27 26 28 23 15 24 25 24 25 23 28 27 27 16 24 30 24 24 2619 28 32 17 25 22 28 27 27 23 20 30 18 26 29 32 28 34 26 24 23 19 28 2533 30 32 27 28 29 20 27 32 28 28 32 28 27 23 21 22 24 29 30 27 20 32 2225 23 32 30 28 24 30 23 30 23 25 23 19 28 23 24 22 28 24 26 23 27 29 2529 29 27 27 26 32 23 26 25 25 28 34 27 30 27 32 27 30 32 28 23 28 24 3328 32 20 29 29 23 23 30 27 24 23 30 23 25 30 28 28 31 28 25 23 19 27 3229 29 26 23 32 33 25 29 27 26 30 34 27 25 34 27 23 35 33 28 32 28 29 3623 24 32 20 23 37 25 24 27 24 38 25 28 28 28 39 29 32 19 27 40 29 33 2332 41 25 28 26 30 42 28 29 27 23 43 24 32 28 29 44 24 25 20 23 45 28 2424 46 32 27 28 47 33 28 27 48 28 30 32 49 29 28 30 50 32 30 23 51 25 3029 52 24 23 23 53 27 26 54 28 27 55 30 34 56 28 32 57 30 32 58 30 27 5923 28 60 26 19 61 27 23 62 34 26 63 32 27 64 32 28 65 27 20 66 28 24 6719 28 68 23 27 69 26 32 70 27 30 71 28 23 72 20 29 73 24 23 74 28 75 2776 32 77 30 78 23 79 29 80 23

[0101] TABLE 8 No. of live mussels in each pot over time when exposed to5 g of Batch 7 (1.15 g KCl) Time C2 C3 C4 C5 C6 C7 C8 C9 C10 C11 (hours)C1 0.5 1 1.5 2 2.5 3 3.5 4 5.5 8.5 1 10 10 7 6 5 4 4 3 1 0 0 2 10 9 4 33 2 1 1 1 0 0 3 10 8 6 4 2 1 1 1 1 1 0 4 10 9 8 7 6 5 4 4 3 2 0 5 10 9 54 3 3 2 2 1 0 0 6 10 10 9 8 6 4 4 3 2 1 0 7 10 8 5 4 4 4 4 2 2 2 0 8 1010 8 8 7 6 5 4 3 3 0

[0102] TABLE 9 No. of live mussels in each pot over time (1.15 g KCl)Time C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 C11 C12 C13 (hrs) 0.5 1 1.5 2 3 3.54.5 5 6 7.5 9 11.5 24 1 10 10 9 9 8 7 7 6 6 3 3 3 0 4 10 10 10 9 9 8 8 77 5 4 4 0 3 10 9 9 7 7 6 6 4 4 3 3 3 0 4 10 10 10 10 10 9 9 8 7 6 3 3 05 10 10 10 10 9 9 8 7 6 6 5 5 0 6 10 9 9 9 8 8 7 6 4 4 3 3 1 7 10 10 109 9 7 5 4 4 3 2 2 0 8 10 10 10 10 9 9 7 5 5 3 2 2 1

[0103] As can be seen in FIG. 9 (mean numbers of mussels alive perbeaker at intervals in the first 24 hours following the five differenttreatments) with 0.5 g and 0.05 g of Batch 7 and with only Palmitic acidadded, there is no mortality within the first 24 hours (although thereis a little mortality with both concentrations of Batch 7 over the nexttwo days. Both 5 g of Batch 7 and 1.15 g of KCl cause rapid mortality,especially over the first 8 hours. Batch 7 seems to be the most rapidlyacting of these two treatments, even though both contain the samequantity of salt.

[0104]FIG. 10 (the mean lengths of remaining mussels at intervals afteraddition of treatment 1) shows that with treatment 1 there aresignificant differences in the sizes of mussels that die at varioustimes in the experiment (1-way anova, df=9, p=0.05).

[0105] Mean sizes of the fatalities and survivors with addition oftreatments 2 and 3 were then compared. FIG. 3 summarises the resultswith the two treatments pooled. The mean size of the survivors issignificantly smaller than that of those that died (two sample t-test,df=158, p=0.0001).

[0106]FIGS. 12 and 13 are scanning electron micrographs (SEMs) ofparticles comprising 90 micron potassium permanganate coated in palmiticacid, the particles being manufactured by fluidised bed spraycongealing. The particles shown are in the size range 150 to 250 microns(as determined by size fractioning by sieving).

[0107]FIGS. 14 and 15 are SEMs of similar particles but of a sizegreater than 250 microns.

[0108]FIG. 16 is a SEM of particles of a size greater than 250 micronscomprising 98 micron potassium chloride coated in palmitic acid,manufactured by fluidised bed spray congealing.

[0109]FIG. 17 comprises a series of photomicrographs of particles fromtwo different samples indicated by (7) and (9), during sizefractionation by sieving (the numbers 90, 150, 250 indicates the size inmicrons of sieve the sample was retained on whereas Res indicates thatthe sample passed through a 90 micron sieve). The bars shown are equalto 400 μm. Sample (7) is the same sample as used in Experiment 8 whereassample (9) comprises 43 μm potassium chloride particles to which 4 g ofsilica was added before spraying with stearic acid for 30 minutes.

[0110] The reader's attention is directed to all papers and documentswhich are filed concurrently with or previous to this specification inconnection with this application and which are open to public inspectionwith this specification, and the contents of all such papers anddocuments are incorporated herein by reference.

[0111] All of the features disclosed in this specification (includingany accompanying claims, abstract and drawings), and/or all of the stepsof any method or process so disclosed, may be combined in anycombination, except combinations where at least some of such featuresand/or steps are mutually exclusive.

[0112] Each feature disclosed in this specification (including anyaccompanying claims, abstract and drawings), may be replaced byalternative features serving the same, equivalent or similar purpose,unless expressly stated otherwise. Thus, unless expressly statedotherwise, each feature disclosed is one example only of a genericseries of equivalent or similar features.

[0113] The invention is not restricted to the details of the foregoingembodiment(s). The invention extends to any novel one, or any novelcombination, of the features disclosed in this specification (includingany accompanying claims, abstract and drawings), or to any novel one, orany novel combination, of the steps of any method or process sodisclosed.

1. Particles for ingestion by an invertebrate organism, said particlesincluding at least one ingredient having desired biological activity inthe organism when ingested by the organism and at least one carriermaterial, characterized in that the active ingredient is solid, toxic tothe organism, and coated with or encapsulated within a water solublecarrier material.
 2. Particles according to claim 1 wherein the activeingredient is embedded throughout the carrier material.
 3. Particlesaccording to any of the preceding claims wherein the organism is anaquatic invertebrate.
 4. Particles according to claim 3 wherein theorganism is a filter feeder.
 5. Particles according to claim 4, whereinthe organism is selected from the group including mussels, clams,oysters and insect larvae.
 6. Particles according to claim 5 wherein theorganism comprises Zebra mussels. (Dreissena polymorpha).
 7. Particlesaccording to any of the preceding claims wherein the active ingredienthas a physiological effect on the organism when ingested by theorganism.
 8. Particles according to claim 1 having a mean particle sizein the range 1 to 200 microns in diameter.
 9. Particles according toclaim 8 having a mean particle size in the range 2 to 150 microns indiameter.
 10. Particles according to any of the preceding claims whereinthe carrier material is nutritious for and/or attractive to the targetorganism, thereby enhancing the ingestion of the particles. 11.Particles according to claim 10 wherein the active ingredient isprovided as a core surrounded by a coating of innocuous and/ornutritious and/or attractive carrier material around said core. 12.Particles according to any of the preceding claims and manufacturedusing complex coacervation, fluidised bed spray coating, or spraycongealing techniques.
 13. Particles according to claim 11 or claim 12wherein the core is between 40 and 60 microns in diameter and thecoating is between 5 and 40 microns.
 14. Particles according to any ofthe preceding claims wherein the active ingredient is an inorganicsolid.
 15. Particles according to claim 14 wherein the active ingredientis potassium chloride or potassium permanganate.
 16. Particles accordingto any of claims 1 to 13 wherein the active ingredient is a saponin. 17.Particles according to claim 16 wherein the active ingredient is derivedfrom Endod berries or is a synthesised form of the active ingredients ofEndod berries.
 18. Particles according to any of the preceding claimswherein the particles are at least partially resistant to water. 19.Particles according to any of the preceding claims wherein the carriermaterial is selected from the group including starch, chocolate, waxes,beeswax, fatty acids, oils, fats or derivatives thereof, and driedplankton.
 20. Particles according to claim 19 wherein the carriermaterial comprises one or more fatty acids such as palmitic acid orstearic acid.
 21. Particles according to any of the preceding claims andhaving neutral buoyancy in freshwater.
 22. Particles according to any ofthe preceding claims and including a surfactant such as sodiumpalmitate.
 23. A method of inducing an invertebrate organism to ingest asubstance having desired biological activity in the organism wheningested by the organism, the method including introducing into theorganism's environment particles ingestible by the organism andincluding the substance and at least one carrier material, wherein thesubstance is solid and coated with or encapsulated within the carriermaterial and wherein the substance is toxic to the target organism. 24.A method according to claim 23 wherein the method includes the step ofcontacting the organisms with the particles for at least 4 hours, morepreferably for between 4 and 8 hours.
 25. A method of concentrating, toa biologically active concentration within an invertebrate organism, atleast one substance, the method comprising providing in the organism'senvironment particles containing the substance and at least one carriermaterial, the particles being ingested by the organism such as to effectsaid concentration of the substance, wherein the substance is solid andcoated with or encapsulated within the carrier material and wherein thesubstance is toxic to the target organism.
 26. A method of preventingcessation of feeding in an invertebrate organism which would otherwiseoccur due to the presence of a substance in the organism's environment,the method including the step of providing the substance in the form ofparticles ingestible by the organism, said particles also including acarrier material, wherein the substance is solid and coated with orencapsulated within the carrier material and wherein the substance istoxic to the target organism.
 27. A method of controlling an invasive orpotentially invasive population of invertebrate organisms, comprisingfeeding said population with an effective amount of a compositioncomprising particles containing a carrier material and at least oneactive ingredient, wherein the active ingredient is solid and coatedwith or encapsulated within the carrier material and wherein the activeingredient is toxic to the target organism.
 28. A method according toclaim 27 wherein an effective amount is a sufficient amount to kill asignificant number of individuals within the population or to reduce thetendency of the population to become invasive.
 29. A method of treatingwater containing invertebrate organisms, comprising adding to the waterparticles containing at least one carrier material and at least oneactive ingredient, said particles being ingestible in an effectiveamount by the invertebrate organisms, wherein the active ingredient issolid and coated with or encapsulated within the carrier material andwherein the active ingredient is toxic to the target organism.
 30. Amethod of controlling parasites or symbionts in or on a hostinvertebrate organism, the method comprising feeding the host organismwith particles including at least one carrier material and at least oneactive ingredient having biological activity in the host and/or in theparasite or symbiont, wherein the active ingredient is solid and coatedwith or encapsulated within the carrier material and wherein the activeingredient is toxic to the target organism.
 31. A method of rendering anenvironment suitable for the farming of invertebrate organisms, themethod comprising adding to the environment particles containing atleast one carrier material and at least one active ingredient, saidparticles being ingestible by the organisms, wherein the activeingredient is solid and coated with or encapsulated within the carriermaterial and wherein the active ingredient is toxic to the targetorganism.
 32. A food product for the farming of invertebrate organisms,the food and product comprising particles ingestible by the organismshaving at least one active ingredient and at least one carrier material,wherein the active ingredient is solid and coated with or encapsulatedwithin the carrier material and wherein the active ingredient is toxicto the target organism.
 33. Particles for ingestion by an invertebrateorganism, said particles including at least one ingredient havingdesired biological activity in the organism when ingested by theorganism and at least one carrier material.